Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism ... [more ▼]

Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism is a crucial issue. Light, carbon and nitrogen availability have been reported to induce important metabolic adaptations. So far, the influence of these variables has essentially been studied while varying only one or two environmental factors at the same time. The goal of the present work was to model the cellular proteomic adaptations of the green microalga Chlamydomonas reinhardtii upon the simultaneous changes of light intensity, carbon concentrations (CO2 and acetate) and inorganic nitrogen concentrations (nitrate and ammonium) in the culture medium. Statistical design of experiments (DOE) enabled to define 32 culture conditions to be tested experimentally. Relative protein abundance was quantified by two dimensional differential in-gel electrophoresis (2D-DIGE). Additional assays for respiration, photosynthesis, and lipid and pigment concentrations were also carried out. A hierarchical clustering survey enabled to partition biological variables (proteins + assays) into eight co-regulated clusters. In most cases, the biological variables partitioned in the same cluster had already been reported to participate to common biological functions (acetate assimilation, bioenergetic processes, light harvesting, Calvin cycle and protein metabolism). The environmental regulation within each cluster was further characterized by a series of multivariate methods including principal component analysis and multiple linear regressions. This metadata analysis enabled to highlight the existence of a clear regulatory pattern for every cluster and to mathematically simulate the effects of light, carbon and nitrogen. The influence of these environmental variables on cellular metabolism is described in details and thoroughly discussed. This work provides an overview of the metabolic adaptations contributing to maintain cellular homeostasis upon extensive environmental changes. Some of the results presented here could be used as starting points for more specific fundamental or applied investigations. [less ▲]

Background: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the impact of hypertriglyceridemia on the mitochondrial, sub-mitochondrial and cellular proteomes in the hepatocytes of a hypertriglyceridemic transgenic mouse model overexpressing the human apolipoproteinC-III. Methods: Quantitative comparative proteomics (2D-DIGE) was carried out in both “low-expressor” (LE) and “high-expressor” (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. Results: The mitoproteome analysis revealed the occurrence of a large-scale adaptation in transgenic mice consisting of a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. Remarkably, the magnitude of these proteomic changes appears to strongly depend on the TG plasma level. Altogether, our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for de novo TG biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes also tend to confirm that the cytoplasm of HTG mice is subjected to oxidative stress as previously stated. The up-regulation of cytosolic ferritin indicates that iron over-accumulation could take place in the cytosol of HE mice hepatocytes and contribute to (i) enhance oxidative stress and (ii) promote cellular proliferation. Conclusions: The present analyses demonstrate that important TG dose-responsive metabolic adaptations are set up in human apolipoproteinC-III-overexpressing mice. Our results indicate that these adaptations could support the higher TG production rates which have been previously reported in this HTG model, and also suggest that cytosolic oxidative stress may result from FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes. [less ▲]

In Chlamydomonas reinhardtii, the Aox1 gene encoding the mitochondrial alternative oxidase (AOX) is tightly regulated by the N source: its transcription is stimulated by nitrate and repressed by ammonium ... [more ▼]

In Chlamydomonas reinhardtii, the Aox1 gene encoding the mitochondrial alternative oxidase (AOX) is tightly regulated by the N source: its transcription is stimulated by nitrate and repressed by ammonium. AOX has been proposed to play a key role in regards to the N source by acting as a trigger factor for the TCA cycle to promote the production of reduced cofactors which, together with photo-generated reductant, could contribute to support nitrate assimilation. In the present work, the cellular proteomes of wild-type and AOX-deficient strains grown either on nitrate or ammonium in the presence of acetate have been compared using two dimensional-differential in-gel electrophoresis (2D-DIGE) coupled to hierarchical clustering and ANOVA-2 statistical analyses in order to further investigate the physiological role(s) of AOX, particularly in regards to the N source. The dramatic up-regulation of chloroplastic components of the carbon-concentrating mechanism (CCM) in response to Aox1 inactivation importantly suggests that AOX plays a key role in the CCM in both N sources by allowing the production of CO2 through the TCA cycle. The concomitant down-regulation of photorespiratory enzymes in cells lacking AOX indicates that such an induction of the CCM could actually be sufficient to over-compensate the effects of AOX deficiency on CO2 availability in the local environment of Rubisco and maintain high rates of inorganic carbon fixation despite lower TCA cycle-mediated CO2 production. The global overexpression of diverse photosynthetic chain and Calvin cycle components in cells lacking AOX further evidences that a tight functional relationship exists between mitochondrial and chloroplastic processes of energy transduction, and that there could be a global redirection of bioenergetics towards photosynthesis to limit the harmful consequences (particularly oxidative stress) of mitochondrial deficiency. The observation that numerous N-responsive proteomic modifications are strongly attenuated (or even abolished) in response to Aox1 inactivation supports that AOX also exerts a more specific role as a central effector of the metabolic plasticity induced by modulating the N source in the growth medium. Our proteomic results tend notably to confirm that respiration (through AOX activity) and photosynthesis collaborate to provide nitrate reduction with electrons in mixotrophic condition and that acetate oxidation could be promoted to support AOX involvement in this context. [less ▲]

Background: In photosynthetic organisms, the influence of light, carbon and inorganic nitrogen sources on the cellular bioenergetics has extensively been studied independently, but little information is ... [more ▼]

Background: In photosynthetic organisms, the influence of light, carbon and inorganic nitrogen sources on the cellular bioenergetics has extensively been studied independently, but little information is available on the cumulative effects of these factors. Here, sequential statistical analyses based on design of experiments (DOE) coupled to standard least squares multiple regression have been undertaken to model the dependence of respiratory and photosynthetic responses (assessed by oxymetric and chlorophyll fluorescence measurements) upon the concomitant modulation of light intensity as well as acetate, CO2, nitrate and ammonium concentrations in the culture medium of Chlamydomonas reinhardtii. The main goals of these analyses were to explain response variability (i.e. bioenergetic plasticity) and to characterize quantitatively the influence of the major explanatory factor(s). Results: For each response, 2 successive rounds of multiple regression coupled to one-way ANOVA F-tests have been undertaken to select the major explanatory factor(s) (1st-round) and mathematically simulate their influence (2nd-round). These analyses reveal that a maximal number of 3 environmental factors over 5 is sufficient to explain most of the response variability, and interestingly highlight quadratic effects and second-order interactions in some cases. In parallel, the predictive ability of the 2nd-round models has also been investigated by k-fold cross-validation and experimental validation tests on new random combinations of factors. These validation procedures tend to indicate that the 2nd-round models can also be used to predict the responses with an inherent deviation quantified by the analytical error of the models. Conclusions: Altogether, the results of the 2 rounds of modeling provide an overview of the bioenergetic adaptations of C. reinhardtii to changing environmental conditions and point out promising tracks for future in-depth investigations of the molecular mechanisms underlying the present observations. [less ▲]

We aimed to modelize the dependence of photosynthetic as well as cytochromial and alternative respiratory maximal activities upon different culture parameters, i.e. light intensity and acetate, carbon ... [more ▼]

We aimed to modelize the dependence of photosynthetic as well as cytochromial and alternative respiratory maximal activities upon different culture parameters, i.e. light intensity and acetate, carbon dioxide, nitrate and ammonium concentrations, in the model unicellular green alga Chlamydomonas reinhardtii. Culture parameters were submitted to a dedicated statistical software (JMP 10.0, SAS Institute) for the generation of a design of experiment. Measurements of oxygen concentrations were carried out to quantify maximal activities. JMP was further used to detect culture parameters exerting a statistically significant effect on maximal activities and to modelize the dependence of maximal activities upon these culture parameters of interest. [less ▲]

In the model unicellular green alga Chlamydomonas reinhardtii, both nitrate and ammonium can be used as primary inorganic nitrogen sources. Interestingly, the expression of the mitochondrial alternative ... [more ▼]

In the model unicellular green alga Chlamydomonas reinhardtii, both nitrate and ammonium can be used as primary inorganic nitrogen sources. Interestingly, the expression of the mitochondrial alternative oxidase (AOX), an "energy-dissipating" ubiquinol-oxygen oxidoreductase of the mitochondrial electron transport chain, is under the control of the exogenous nitrogen source : it is activated in nitrate-grown cells and repressed in ammonium-grown cells at both transcriptional and translational levels. This regulation of AOX by nitrogen is Chlamydomonas-specific and currently its bioenergetic and metabolic significance is poorly understood. In order to get clues to this peculiar phenomenon, we characterized the global metabolic response of a wild-type strain (WT) and an AOX-deficient mutant (AOX-) obtained by RNA interference grown either on nitrate or ammonium. For this purpose, we used a highly accurate 2D electrophoresis-based comparative proteomic approach (2D-DIGE) to compare the cellular proteomes of nitrate and ammonium-grown WT and AOX- Chlamydomonas. The analysis was performed in the middle of the exponential growth phase in mixotrophic conditions. It revealed many proteomic modifications between WT and AOX- cells and a smaller number between nitrate and ammonium-grown cells. In nitrate-grown cells, we notably observed an important up-regulation of glutamine synthetase. Interestingly, in AOX- cells, we respectively detected a general down-regulation and a general up-regulation of mitochondrial and chloroplastic bioenergetic enzymes, and also an important up-regulation of glutathione-dependent oxidative stress defense systems together with a remarkable down-regulation of methionine synthase. Altogether these results and previous studies provide new features in understanding the metabolic adaptations occurring in response to the inorganic nitrogen source with emphasis on the role played by AOX. [less ▲]

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase ... [more ▼]

In the present work we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase (AOX). The AOX-deficient strain displays a doubling of the cell volume and biomass without any alteration of the generation time, a significantly higher ROS production, no change in total respiration rate, and a slight decrease of the photosynthesis efficiency. In order to identify the molecular adaptation underlying these phenotypical effects, we carried out a comparative proteomic study at the level of the mitochondrial and cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways. [less ▲]

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one ... [more ▼]

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c1 together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster. [less ▲]